Pharmaceutical compositions comprising attenuated plasmodium sporozoites and glycolipid adjuvants

ABSTRACT

Disclosed herein are pharmaceutical compositions comprising  Plasmodium  sporozoite-stage parasites and compatible glycolipid adjuvants useful in vaccines for preventing or reducing the risk of malaria. In particular, human host range  Plasmodium  and analogues of α-galactosylceramide (α-GalCer), a ligand for natural killer T (NKT) cells, are combined in pharmaceutical compositions, which are useful as vaccines against malaria. Methods of use are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from the U.S. Provisional application61/485,092, filed May 11, 2011, the contents of which are herebyincorporated by reference their entireties.

FIELD OF THE INVENTION

The invention disclosed herein relates generally to pharmaceuticalcompositions comprising Plasmodium parasites useful as immunogens invaccines for preventing or reducing the risk of malaria. Moreparticularly, the invention relates to pharmaceutical compositionscomprising Plasmodium sporozoite-stage parasites and glycolipidadjuvants wherein the compositions are useful for eliciting an immuneresponse, conferring protective immunity in a host so as to preventmalaria, and reducing the incidence of malaria in hosts subsequentlychallenged with pathogenic Plasmodium, more particularly in mammalian orhuman hosts, and the invention relates to vaccines and methods of usingthe provided pharmaceutical compositions in vaccines for the preventionof malaria.

BACKGROUND OF THE INVENTION

There are >200 million malaria cases and ˜1 million deaths per yearcaused by Plasmodium falciparum (N) (World Health Organization. GlobalMalaria Programme. World Malaria Report 2010: World Health Organization,2010; Murray C J, et al. Global malaria mortality between 1980 and 2010:a systematic analysis. 2012 Lancet 379: 413-431). Recently, progress hasbeen made controlling malaria due to investment of billions of dollarsin the use of bednets, insecticides, and drugs. However, as highlightedin a recent editorial (Editorial: Malaria 2010: More Ambition andAccountability Please 2010 Lancet 375:1407), a commercially availablemalaria vaccine is still badly needed. To prevent infection, disease,and transmission an ideal single stage vaccine should target thepre-erythrocytic (sporozoite and liver) stages (Plowe C. V. et al. Thepotential role of vaccines in the elimination of falciparum malaria andthe eventual eradication of malaria 2009 J. Infect. Dis. 200:1646-1649;Alonso P. L. et al. A research agenda for malaria eradication: vaccines.2011 PLoS Med 8: e1000398). Such a vaccine would have hμge public- andprivate-sector markets. In public-sector markets it would be used ininfants, young children, and adolescent females (preventing malariaduring pregnancy) and for entire populations for geographically focusedmalaria elimination campaigns (Id.). Individuals from non-malariouscountries who spend time in areas with malaria (travelers, military,government officials, students, business people, etc.) and middle andupper class residents of countries with malaria comprise theprivate-sector market.

Data indicating a highly effective vaccine might be possible came fromtrials in which volunteers immunized by the bites of mosquitoes infectedwith radiation-attenuated Pf sporozoites had high-level (>90%),sustained (≧10 months) protection against experimental challenge(Hoffman, S. L., et al. 2002 J. Inf. Dis. 185:1155-64).

It has been shown that a vaccine incorporating live attenuated Pfsporozoites can be manufactured (SANARIA™ PfSPZ Vaccine). This processhas recently been described (Hoffman S. L. et al. Development of ametabolically active, non-replicating sporozoite vaccine to preventPlasmodium falciparum malaria. 2010 Hum. Vac. 6:97-106. DOI: 10396[pii].). The ability of PfSPZ Vaccine to induce antigen-specific immuneresponses in humans was also demonstrated (Epstein, J. E., et al. LiveAttenuated Malaria Vaccine Designed to Protect Through Hepatic CD8⁺ TCell Immunity 2011 Science 334 (6055):475-480).

It is thought that an attenuated PfSPZ vaccine delivered by a parenteralnon-intravenous route and capable of demonstrating a protective efficacycomparable to that achieved with PfSPZ administered IV would requirelarge numbers of sporozoites and a multi-dose regimen. In a mouse model,present data suggests that approximately 7 times as many Plasmodiumyoelii (Py) sporozoites administered intradermal (ID) or subcutaneous(SC) are required compared to IV administration in order to achieve >80%protection in mice (Epstein, et al., Id.). A more promising approach forthe development of a highly effective parenteral non-IV vaccine wouldlikely include the use of an adjuvant.

The adjuvant: A glycolipid adjuvant that stimulates natural killerT-cells (NKT) was identified in mice. (Gonzalez-Aseguinolaza G, et al.Natural killer T cell ligand α-galactosylceramide enhances protectiveimmunity induced by malaria vaccines 2002 J. Exp. Med. 195: 617-624;U.S. Pat. No. 7,534,434). Using a single IV-administered dose ofradiation attenuated P. yoelii sporozoites (suboptimal for protection)it was demonstrated in the Gonzalez-Aseguiniola paper that distalintraperitoneal (IP) administration of α-galactosylceramide (α-GalCer),a ligand for natural killer T (NKT) cells, could induce a higher degreeof protection (>90%), than IV administration of irradiated P. yoeliisporozoites alone, which conferred only 20% protection.

Natural Killer T (NKT) cells are a subset of T cells that co-expressreceptors of T cell and NK cell lineages and recognize their cognateantigen presented by the MHC-like CD1d on antigen presenting cells(APCs). The major subset of NKT cells are distinguished by theirrestricted expression of an invariant TCR (invTCR) and are termed iNKTcells. The increased potency of IV administered sporozoites and distallyadministered IP adjuvant described in Gonzalez-Aseguinolaza et al.correlated with enhanced IFN-gamma secretion by CD8⁺ T cells and wasdependent on iNKT cells and CD1d (Id.). This first-identified iNKT TCRligand, α-GalCer, extracted from the Agelas mauritianus sea sponge, wasdiscovered while screening for compounds with anti-tumor activity. Ithas a high affinity for CD1d, is a potent activator of iNKT cells inboth mouse and human, and has been used extensively to study thefunction of iNKT cells (Brossay L, et al. CD1d-Mediated Recognition ofan A-Galactosylceramide by Natural Killer T Cells is Highly Conservedthrough Mammalian Evolution 1998 J. Exp. Med. 188:1521-1528; KobayashiE, et al. KRN7000, A Novel Immunomodulator, and its Antitumor Activities1995 Oncol. Res. 7: 529-534; Kawano T, et al., CD1d-restricted andTCR-mediated activation of vα14 NKT cells by glycosylceramides 1997Science 278: 1626-1629. In vivo administration of α-GalCer in miceresults in a cascade of events beginning with signaling through theinvTCR by APCs expressing CD1d. Macrophages, dendritic cells, B cells,Kupffer cells in the liver, and hepatocytes all have constitutiveexpression of CD1d (Mandal M, et al., Tissue distribution, regulationand intracellular localization of murine CD1 molecules 1998 Mol.Immunol. 35: 525-536; Brossay L., et al., Mouse CD1 is mainly expressedon hemopoietic-derived cells 1997 J. Immunol 159: 1216-1224; Roark, J.H., et al., A. CD1.1 expression by mouse antigen-presenting cells andmarginal zone B cells 1998 J. Immunol. 160: 3121-3127).

Stimulated iNKT cells rapidly secrete pre-stored cytokines (unliketraditional T cells) that reciprocally activate APCs (Tomura M., et al.,A novel function of Vα14+CD4+NKT cells: stimulation of IL-12 productionby antigen presenting cells in the innate immune system 1999 J. Immunol.163: 93-101; Fujii, S., et al., Activation of natural killer T cells byα-galactosylceramide rapidly induces the full maturation of dendriticcells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 Tcell immunity to a coadministered protein 2003 J. Exp. Med. 198:267-279) enhancing their ability to prime CD4⁺ and CD8⁺ T cells (Fujii,S., et al., The linkage of innate to adaptive immunity via maturingdendritic cells in vivo requires CD40 ligation in addition to antigenpresentation and CD80/86 costimulation 2004 J. Exp. Med. 199: 1607-1618;Hermans, I. F., et al., NKT cells enhance CD4+ and CD8+ T cell responsesto soluble antigen in vivo through direct interaction with dendriticcells 2003 J. Immunol. 171: 5140-5147) to generate a powerfulcell-mediated immune response. In the paper of Gonzales-Aseguinolaza etal., supra, the legend of FIG. 2A states that a group of BALB/c mice wasimmunized subcutaneously with irradiated sporozoites [P. yoelii]together with or without administration of α-GalCer by the same route,and when splenic lymphocytes were isolated and the number ofIFN-γ-secreting CS-specific CD8⁺ and CD4⁺ T-cells were determined byELISPOT assay it was found that co-administration of α-GalCer increasedthe number of IFN-γ-secreting CS-specific CD8⁺ cells seven fold. Thus,the overall amplification of the adaptive immune response by iNKT cellsmade them very attractive adjuvant targets.

Subsequently, α-GalCer has been demonstrated to have adjuvant propertiesfor influenza, HIV, and tumor vaccines in mice (Huang, Y., et al.,Enhancement of HIV DNA vaccine immunogenicity by the NKT cell ligand,α-galactosylceramide 2008 Vaccine 26:1807-1816; Ko, S. Y., et al.,α-Galactosylceramide can act as a nasal vaccine adjuvant inducingprotective immune responses against viral infection and tumor 2005 J.Immunol. 175: 3309-3317; Seino, K., et al., Natural killer Tcell-mediated antitumor immune responses and their clinical applications2006 Cancer Sci. 97: 807-812).

Furthermore, because α-GalCer was discovered while screening forcompounds with anti-tumor properties, it has been used in severalclinical trials in cancer patients. Delivery by pre-loading autologousPBMCs with α-GalCer in vitro or by direct injection, α-GalCer was shownto be safe and well tolerated. However, although modest enhancement ofimmune responses was generally seen, its beneficial effects were limited(Giaccone, G., et al., A phase I study of the natural killer T-cellligand α-galactosylceramide (KRN7000) in patients with solid tumors 2002Clin. Cancer Res. 8:3702-3709; Ishikawa, A., et al., A phase I study ofα-galactosylceramide (KRN7000)-pulsed dendritic cells in patients withadvanced and recurrent non-small cell lung cancer 2005 Clin. Cancer Res.11: 1910-1917; Nieda, M., et al., Therapeutic activation of Vα24+Vbeta1+NKT cells in human subjects results in highly coordinated secondaryactivation of acquired and innate immunity 2004 Blood 103: 383-389).

Consequently, Tsuji and colleagues made an effort to find analogues ofα-GalCer with increased CD1d-binding and iNKT-stimulatory properties.The particular advantages of identifying a new glycolipid adjuvantsimilar to α-GalCer and based on a CD1d-binding, iNKT-stimulatory effectare multi-fold. First, the phenotype and functional properties of theCD1d molecules and invTCR of iNKT cells have been conserved betweenhumans and mice, thereby allowing prediction of the activity of relatedglycolipids in humans through mouse studies. Second, α-GalCer itself hasbeen approved and well characterized in terms of safety and activity inhumans. Third, related glycolipids used as vaccine adjuvant could beadministered in much smaller quantities using a local parenteral routeof administration (e.g. intramuscular) than the larger doses of α-GalCercurrently dispensed IV for cancer therapy, thereby further minimizingpotential systemic side effects.

Tsuji and colleagues screened a library of synthetic α-GalCer analoguesand identified glycolipids with far greater CD1d binding and activationof iNKT cells (Li, X., et al., Design of a potent CD1d-binding NKT cellligand as a vaccine adjuvant 2010 Proc Natl Acad Sci USA 107:13010-13015; U.S. Pat. No. 7,923,013). One such glycolipid, was 7DW8-5(FIG. 1). Structurally, 7DW8-5 possesses a fluorinated benzene ring atthe end of C10 length fatty acyl chain. It was selected due to itssuperior ability to elicit cytokine production from human and mouse iNKTcells and its adjuvant properties in mice when used in combination witha suboptimal dose of a recombinant adenovirus expressing P. yoelii CSprotein. The adjuvant was co-administered intramuscularly (IM) with thevaccine. The mice were challenged with pathogenic P. yoelii sporozoites2 weeks later. 7DW8-5 enhanced the malaria-specific CD8⁺ T cell responsesignificantly more than α-GalCer and also enhanced the malaria-specifichumoral response equally if not slightly stronger than α-GalCer.Finally, 7DW8-5 was able to display a significantly stronger adjuvanteffect than α-GalCer in enhancing protective efficacy of the adenovirusrecombinant vaccine after a single immunizing dose.

The practical considerations for the preclinical and clinicaldevelopment of 7DW8-5 as an adjuvant for candidate recombinant subunitmalaria vaccines was recently discussed (Padte, N. N., et al., Clinicaldevelopment of a novel CD1d-binding NKT cell ligand as a vaccineadjuvant 2010 Clin. Immunol. doi: 10.1016/j.clim.2010.11.009).

There is a need for improved malaria vaccines. With regard to malariavaccines whose immunogen is live attenuated Plasmodium parasites,particularly sporozoite-stage parasites, an adjuvant that could reducethe numbers of doses and the dosages of each dose required for highlyeffective protection would have enormous value in the fight againstmalaria. For instance, a vaccine administered in 1 or 2 doses would notonly reduce the cost of goods to produce it, but more importantly itwould simplify the logistics of delivery for travelers, for rapidlydeployed military, or for mass-immunization campaigns.

SUMMARY OF THE INVENTION

Disclosed herein are pharmaceutical compositions comprising one or morespecies of live, Plasmodium sporozoite-stage parasites, a glycolipidadjuvant, and an excipient.

Also disclosed are methods of using these pharmaceutical compositionsfor reducing the risk of malaria in an individual and reducing theincidence of malaria among a group of individuals exposed to pathogenicPlasmodium parasites. These methods comprise administration of 1 or moredoses of the pharmaceutical compositions provided herein prior to saidexposure.

Also disclosed are improved malaria vaccines whose immunogen compriseseither live attenuated Plasmodium sporozoites, or live non-attenuatedsporozoites delivered along with the protection of an anti-malarial drugsuch as chloroquine, each of which being useful for the prevention ofmalaria. Compositions of live Plasmodium sporozoites and the glycolipidadjuvants disclosed herein are compatible in pharmaceutical compositionsand surprisingly effective in reducing the number of doses and/orreducing the effective sporozoites dosage.

DESCRIPTION OF THE DRAWINGS

FIG. 1—Depicts the structures of α-GalCer and 7DW8-5.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “about” or “approximately” means within one standard deviationas per the practice in the art.

“Additive” as used herein as a noun is a compound or composition addedto a sporozoite preparation to facilitate preservation of thepreparation. Additives may include cryoprotectants such as DMSO andglycerol, antioxidants, and the like.

“Conferring protective immunity” as used herein refers to providing to apopulation, a group of individuals or a host (i.e., an individual), theability to generate an immune response which in turn protects against,i.e. prevents, a disease, malaria in this case, caused by a pathogen(e.g. Plasmodium falciparum) such that the clinical manifestations,pathology, or symptoms of disease in a host exposed to the pathogen arereduced as compared to a non-treated host, or such that the rate atwhich infection, or clinical manifestations, pathology, or symptoms ofdisease appear within a population are reduced, as compared to anon-treated population, group or individual.

“Immune response” as used herein means a response in the recipient tothe introduction of attenuated sporozoites generally characterized by,but not limited to, production of antibodies and/or T cells. Generally,an immune response may be a cellular response such as induction oractivation of CD4+ T cells or CD8+ T cells specific for Plasmodiumspecies epitopes, a humoral response of increased production ofPlasmodium-specific antibodies, or both cellular and humoral responses.With regard to a malaria vaccine, the immune response established by avaccine comprising sporozoites includes but is not limited to responsesto proteins expressed by extracellular sporozoites or other stages ofthe parasite after the parasites have entered host cells, especiallyhepatocytes and mononuclear cells such as dendritic cells and/or tocomponents of said parasites. In the instant invention, upon subsequentchallenge by infectious organisms, the immune response prevents orexhibits development of pathogenic parasites to the asexual erythrocyticstage that causes disease.

“Intravenous” (abbreviated as IV) as defined herein means intentionalintroduction, directly into the lumen of an identified large bloodvessel such as a vein.

“Metabolically active” as used herein means alive, and capable ofperforming sustentative functions and some life-cycle processes. Withregard to attenuated sporozoites this includes sporozoites that arecapable of invading hepatocytes in culture and in vivo, progressingthrough some developmental stages within hepatocytes, and displaying denovo expression of stage-specific proteins.

“Parenteral” as defined herein means not through the alimentary canal,but rather by introduction through some other route, as intradermal(ID), subcutaneous (SC), intramuscular (IM), intraperitoneal (IP),intravenous (IV), and the like.

“Pharmaceutical composition” as defined herein means a sterilecomposition comprising one or more active ingredients, producedaceptically, under cGMP protocol, such that the composition would beacceptable for use in human subjects.

“Therapeutic” as defined herein relates to reduction of clinicalmanifestations or pathology which have already become manifest. A“therapeutically effective amount” as used herein means an amountsufficient to reduce the clinical manifestations, pathology, or symptomsof disease in an individual, or an amount sufficient to decrease therate at which clinical manifestations, pathology, or symptoms of diseaseappear within a population.

“Vaccine” as used herein is a preparation comprising an immunogenicagent and a pharmaceutically acceptable diluent in combination withexcipient, adjuvant and/or additive or protectant. The immunogen may becomprised of a whole infectious agent or a molecular subset of theinfectious agent (produced by the infectious agent, or producedsynthetically or recombinantly). When the vaccine is administered to asubject, the immunogen stimulates an immune response such that, uponsubsequent challenge with infectious agent, will protect the subjectfrom illness or mitigate the pathology, symptoms or clinicalmanifestations caused by that agent. A therapeutic (treatment) vaccineis given after infection and is intended to reduce or arrest diseaseprogression. A preventive (prophylactic) vaccine is intended to preventinitial infection or reduce the rate or burden of the infection. Agentsused in vaccines against a parasitic disease such as malaria may bewhole-killed (inactive) parasites, live-attenuated parasites (unable tofully progress through their life cycle), live, fully infectiousparasites administered with an anti-malarial drug, or purified orartificially manufactured molecules associated with the parasite e.g.recombinant proteins, synthetic peptides, DNA plasmids, and recombinantviruses or bacteria expressing Plasmodium proteins. A vaccine maycomprise sporozoites along with other components such as excipient,diluent, carrier, anti-malarial drug, preservative, adjuvant or otherimmune enhancer, or combinations thereof, as would be readily understoodby those in the art.

Chemical nomenclature as used herein is as follows: “R” represents avariable chemical structure attached at the indicated location in afamily of compounds; “Me” represents a methyl group attached at theindicated location; “Ph” represents a phenolic ring attached at theindicated location; “(p-OMe)” or “(p-F)” or “(p-CF₃)” following Phrepresents the corresponding moiety located at the para position of thephenolic ring; “F” represents a fluoride moiety located attached at theindicated location; “O” represents an oxygen moiety, “C” represents acarbon moiety; and “H” represents a hydrogen moiety.

Pharmaceutical Compositions Comprising Plasmodium Sporozoites

In addition to the live attenuated PfSPZ Vaccine described above, thereis another approach to whole sporozoite malaria vaccine developmentcalled chemoprophylaxis with sporozoites (CPS), and requires the bite ofonly 45 mosquitoes (3 times 15 mosquitoes) carrying fully infectiousPfSPZ of volunteers taking chloroquine chemoprophylaxis to achieve 95%sterile protective immunity (19/20 volunteers) and that lasts for atleast 28 months (Roestenberg M, et al. Protection Against a MalariaChallenge by Sporozoite Inoculation. 2009 N. Eng. J. Med. 361: 468-477;Roestenberg M, et al. Long-term Protection Against Malaria AfterExperimental Sporozoite Inoculation: an Open-label Follow-up Study. 2011Lancet 377: 1770-1776). This is 20-25 times fewer PfSPZ-infectedmosquitoes than the 1,000 required for the irradiated PfSPZ approach(Hoffman S L, et al. Protection of humans against malaria byimmunization with radiation-attenuated Plasmodium falciparum sporozoites2002 J. Infect. Dis. 185:1155-1164). As disclosed in Roestenberg 2009,incorporated herein by reference, the antimalarial drug (e.g.,chloroquine) is administered to the host prior to the firstadministration of Plasmodium immunogen in the vaccination regimen,usually at least 2 days prior to first dose, and administration of theantimalarial continues, usually for at least 30 days subsequent to thelast dose in the regimen, such that the level of antimalarial in thebloodstream of said host is sufficient to prevent the signs, symptomsand pathology of malaria. For example, as disclosed in Roestenberg(Id.), chloroquine may be administered orally in two 300 mg dosesstarting 2 days prior to the first exposure to SPZ and continuing inweekly 300 mg doses until 1 month after the last exposure. It is atransformative approach to human vaccination because it harnesses theinfectious agent's inherent replicative properties to amplify productionof protective immunogens spanning multiple developmental stages, andthen eliminates the infectious agent with an anti-infective drug beforethe onset of disease. For either vaccine, an adjuvant that increases thepotency of the SPZ sporozoite would be of enormous value.

Most of the technical hurdles in the development of malaria vaccinescomprising pharmaceutical compositions of live attenuated sporozoitesand live infectious sporozoites have now been overcome—among them,aseptic production of sufficient quantities of sporozoites isolated fromattendant material using cGMP protocol (See particularly U.S. Pat. No.7,229,627; Hoffman, S L, et al., 2010 Hum. Vac. 6:97-106—both explicitlyincorporated herein by reference).

Regarding a vaccine suitable for routine use in human subjects thatcomprises live attenuated sporozoites, and live infectious sporozoites,the sporozoites must be substantially purified from the source fromwhich they were produced. Pharmaceutical compositions comprisingsubstantially purified sporozoites and excipient as well as methods ofpurifying sporozoites are known in the art (U.S. Patent Publ. No.2010/0183680. This publication is explicitly incorporated herein byreference.

Plasmodium-species parasites are grown aseptically in cultures as wellas in vivo in Anopheles-species mosquito hosts, most typically Anophelesstephensi hosts. Methods of axenically culturing Plasmodium-speciesliver stage parasites (Kappe et al. US Pub. 2005/0233435) and methods ofproducing attenuated and non-attenuated Plasmodium-species sporozoites,particularly, methods of growing and attenuating parasites inmosquitoes, and harvesting attenuated and non-attenuated sporozoites areknown in the art and have been described (See: U.S. Pat. No. 7,229,627;U.S. Pub. No. 2005/0220822).

PfSPZ Vaccine, a malaria vaccine comprising live attenuated Plasmodiumsporozoites without adjuvant, has been developed and is in clinicaltrials (Hoffman S. L. et al., Development of a metabolically active,non-replicating sporozoite vaccine to prevent Plasmodium falciparummalaria. 2010 Hum. Vac. 6:97-106. DOI: 10396 [pii].). The vaccine hasalready been assessed in a first-in-humans Phase 1 clinical trial in 80healthy, malaria-naive adults at Naval Medical Research Center and theUniversity of Maryland (Epstein J. E., et al. Supra.). The vaccine wasadministered intradermally (ID) or subcutaneously (SC) to 80 volunteerswith the primary goal of establishing safety. Results of thisdose-escalation study demonstrated the PfSPZ Vaccine is safe, welltolerated, and without breakthrough infections. Furthermore, the vaccineinduced antibody and T cell immune responses, and protected severalvolunteers. However, as expected based on pre-clinical data, optimalimmune responses and protection were not achieved in this first trialand studies in mice, rabbits, and monkeys have pointed the way towardthe next steps in the R&D process (Epstein, J. E. et al, Id.). The Pfsporozoites in the PfSPZ Vaccine are highly potent. Based on animalstudies, it appears that the effectiveness of pre-erythrocytic stagemalaria vaccines correlates with the induction of interferon gammaproducing CD8+ T cells in the liver. It is expected that this immuneresponse will eliminate the Pf-infected liver cells. When rhesus monkeyswere immunized intravenously (IV) with the PfSPZ Vaccine, 4 months afterthe last dose, 3% of all CD8+ T cells in the liver were specific forPfSPZ. No such responses were seen in monkeys immunized by the SC route,as was done in the first clinical trial. In mice, IV administration ofirradiated, purified, cryopreserved P. yoelii SPZ induced high levels ofprotection at low doses; 88% (21/24) of mice were fully protected after3 doses of 2,000 irradiated PySPZ. When administered SC or ID, as in thefirst clinical trial, immune responses were 50 to 150 fold lower and itrequired 7 to 10 times as many sporozoites to achieve high levelprotection.

It is anticipated that IV administration of the vaccine will reduce thenumber of sporozoites required to provide protection against subsequentchallenge with pathogenic parasites and effectively reduce the incidenceof malaria among a group of individuals exposed to pathogenic Plasmodiumparasites. However, even in mice, it currently requires 3 dosesadministered IV to achieve high level protection, and it would bepreferable to achieve such protection with fewer doses. Furthermore,administration by a parenteral non-IV route would be preferable.Therefore, it is desirable to reduce the number of sporozoites per dose,and perhaps the number of doses, required to confer protective immunityby a parenteral non-IV route of administration. These requirementsprovide the framework for identifying an effective adjuvant that iscompatible for use with live attenuated sporozoites.

Prior to the discovery disclosed herein, no adjuvant had been describedas compatible with live attenuated Plasmodium sporozoites. Thesuccessful development of a sporozoite-compatible adjuvant to maximizethe protective efficacy of parenterally administered sporozoites isuniversally applicable to sporozoites attenuated by all means, includingradiation, chemicals or genetic alteration, thereby enabling a highlyeffective vaccine for the prevention of malaria delivered by aparenteral route (IV or non-IV).

Methods of Making Glycolipids

The methods of synthesis of the glycolipid analogues of the presentinvention are provided in Tsuji et al (U.S. Patent Publ. No.2007/02388871, explicitly incorporated by reference). Unlike laboratoryreagents for use in small animal studies, all biologic substances mustbe manufactured under Good Manufacturing Practice (GMP) conditions (Codeof Federal Regulations Title 21, Part 211, April 2009) before use inhumans One of the practical advantages of glycolipids is theirstraightforward synthesis pathway from chemical compounds that arereadily available at relatively low cost. In order for glycolipids tofunction as effective adjuvants that provide dose-sparing of vaccinesagainst infectious diseases with high prevalence in the developingworld, low cost and ease of manufacturing are desirable (Padte, et al.,at page 4).

Five analogues of α-GalCer were chosen based on enhanced stimulatoryactivity against human iNKT cells in vitro, binding affinity to humanand murine CD1d molecules, and binding affinity to the invariant t cellreceptor of human iNKT cells (Li, X., et al., Design of a potentCD1d-binding NKT cell ligand as a vaccine adjuvant 2010 Proc Natl AcadSci USA 107: 13010-13015, explicitly incorporated herein by reference).

The genus of the 5 analogues is shown schematically in formula (1):

where R is: (CH₂)₅Ph(p-OMe); (CH₂)₇Ph(p-OMe); (CH₂)₇Ph(p-F);(CH₂)₁₀Ph(p-F); or (CH₂)₁₀Ph(p-CF₃).

Each of these 5 analogues are designated as follows:

formula (2) (designated as C18 in Li et al., and designated formula 101in U.S. Pat. No. 7,923,013)

formula (3) (designated as C22 in Li et al., and designated as formula102 in U.S. Pat. No. 7,923,013)

formula (4) (designated as C23 in Li et al., and designated as formula104 in U.S. Pat. No. 7,923,013)

formula (5) (designated as 7DW8-5 in Li et al., and designated asformula 105 in U.S. Pat. No. 7,923,013)

formula (6) (designated as 7DW8-6 in Li et al., and designated asformula 108 in U.S. Pat. No. 7,923,013)

Administration

In certain embodiments, a pharmaceutical composition comprising one ormore species of live Plasmodium sporozoite-stage parasites isco-administered with a glycolipid adjuvant. In some embodiments, theco-administration is by the same or a different route of administration.For example, a pharmaceutical composition comprising one or more speciesof live Plasmodium sporozoite-stage parasites administered by anintravenous, intramuscular, intradermal, or subcutaneous route can beco-administered with a glycolipid adjuvant administered by anintravenous, intramuscular, intradermal, or subcutaneous route. In afurther embodiment, an antimalarial drug can be further co-administeredby an intravenous, intramuscular, intradermal, or subcutaneous route.

In some embodiments, the co-administration is concurrent, e.g., as anadmixture. In some embodiments, the co-administration is sequential. Incertain embodiments, the time between sequential administration eventsis not greater than about one hour, not greater than about 30 minutes,not greater than about 15 minutes, not greater than about 10 minutes, ornot greater than about 5 minutes between the administrations. In certainembodiments, the time between co-administration events is 0-60 minutesbetween administrations, 0-30 minutes between administrations, 0-15minutes administrations, 0-10 minutes between administrations, or 0-5minutes between administrations.

In certain embodiments, a pharmaceutical composition comprising one ormore species of live Plasmodium sporozoite-stage parasites isco-administered with one or more glycolipid adjuvants and additionallyin the presence of an antimalarial drug at sufficient concentration inthe bloodstream of the host to prevent the signs, symptoms or pathologyof malaria upon subsequent exposure to pathogenic parasites. In otherembodiments, a pharmaceutical composition comprising one or more speciesof live Plasmodium sporozoite-stage parasites and a glycolipid adjuvantare administered on the same time course or administered on anoverlapping time course relative to an antimalarial drug, i.e., theantimalarial drug is not co-administered. As described, in someembodiments, the antimalarial drug was previously administered such thatthe concentration of said drug in the bloodstream of said host issufficient to prevent the clinical manifestations of malaria.

Pharmaceutical Compositions

In compiling the results of the experiments disclosed in Examples 1through 3, a combined 13% of BALB/c mice inoculated with radiationattenuated P. yoelii sporozoites (irr PySPZ) were protected fromsubsequent challenge by immunization with one dose administered IV. Thislevel of protection improved to 75% when 7DW8-5 (formula (5)) was firstdelivered to the subject mice by intraperitoneal (IP) injection followedby the suboptimal IV dose of radiation attenuated Py sporozoites. Thesedata show that 7DW8-5, administered distally and IP, can reduce thenumber of doses required to confer protection.

TABLE 1 Summary of experiments described in Examples 1-3 protected/percent Regimen challenged protected irr PySPZ delivered IV 4/30 13% irrPySPZ delivered IV + 30/40  75% 7DW8-5 delivered IP 7DW8-5 delivered IP0/15 0% Infectivity controls 0/23 0%

While P. yoelii is a species of Plasmodium with a mouse host range, itis widely used and generally considered a predictive model of thebehavior of human host range Plasmodium by those skilled in the art (Seee.g. Khan, Z. M. and J. P. Vanderberg (1991) Role of Host CellularResponse in Differential Susceptibility of Nonimmunized BALB/c Mice toPlasmodium berghei and Plasmodium yoelii Sporozoites. Infect. and Immun.59(8):2529-2534). Plasmodium species with human host range include P.falciparum, P. vivax, P. ovale, P. knowlesi, and P. malariae.

All of the references cited above, as well as all references citedtherein, are incorporated herein by reference in their entireties.

EXAMPLES Example 1 Immunization of BALB/c Mice with Fresh UnpurifiedRadiation Attenuated Plasmodium yoelii Sporozoites

To determine if the adjuvant 7DW8-5 could decrease the number of dosesof irr PySPZ required to confer protection, a suboptimal dosing regimenwas performed. irr PySPZ (17NXL) were dissected from the salivary glandsof A. stephensi mosquitoes and injected intravenously (IV) into BALB/cmice as a single dose of either of 5×10⁴ or 1×10⁴ sporozoites. Two μg of7DW8-5 was concurrently administered intraperitoneally (IP) in somemice. Fourteen days later, mice were challenged with 100 non-irradiatedpathogenic P. yoelii sporozoites. Protection was assessed byGiemsa-stained blood smears and was defined as the complete absence ofparasitemia 7 and 14 days post infection.

In the absence of adjuvant, 0/5 mice and 1/5 mice that received 5×10⁴and 1×10⁴ respectively were protected. When administered with adjuvant,3/5 and 4/5 mice were protected, respectively. Adjuvant alone providedno protection, indicating an antigen specific response.

TABLE 2 Example #1. Immunization with fresh, unpurified radiationattenuated Py sporozoites # of BALB/c fresh, unpurified irr 2 μg#protected/ mice PySPZ IV 7DW8-5 IP #Challenged 5 5 × 10⁴ − 0/5 5 5 ×10⁴ + 3/5 5 1 × 10⁴ − 1/5 5 1 × 10⁴ + 4/5 5 — + 0/5 5 Infectivitycontrols − 0/5

Example 2 Immunization of BALB/c Mice with Fresh Unpurified RadiationAttenuated Py Sporozoites

A second experiment was performed to confirm the results shown inExample 1. Using the same method as Example 1, a single dose of 1×10⁴irr PySPZ did not protect any of 10 mice, but when combined withadjuvant protected 7 of 10 mice. Again, adjuvant in the absence ofantigen had no effect.

TABLE 3 Example #2. Immunization with fresh, unpurified radiationattenuated Py sporozoites # of fresh, unpurified irr 2 μg #protected/BALB/c mice PySPZ IV 7DW8-5 IP #challenged 10 1 × 10⁴ − 0/10 10 1 ×10⁴ + 7/10 5 — + 0/5  10 Infectivity control − 0/10

Example 3 Immunization of BALB/c Mice with Fresh Purified RadiationAttenuated Py Sporozoites

Using the single dose methodology of Example 1 with radiation attenuatedPy sporozoites that have been purified using the purification proceduredisclosed in Sim et al. (U.S. Pat. No. 8,043,625, incorporated in itsentirety herein by reference) a third experiment was performed. Using1×10⁴ purified sporozoites administered IV in a single dose, 3 out of 10mice were protected, whereas 7 of 10 mice were protected when theadjuvant was concurrently administered IP.

TABLE 4 Example #3. Immunization with purified radiation attenuated Pysporozoites number of BALB/c fresh irr PySPZ 2 μg # protected/ mice IV7DW8-5 IP # challenged 10 1 × 10⁴ + 9/0 unpurified 10 1 × 10⁴ −  3/10purified 10 1 × 10⁴ +  7/10 purified 5 — + 0/5 8 Infectivity controls −0/8

Example 4 Rechallenge—Longevity of Protection

Fifteen weeks after their first challenge with fresh, infectious P.yoelii sporozoites, the mice that had received a single dose of 10⁴irradiated P. yoelii sporozoites with or without concurrentadministration of adjuvant IP and were protected in Experiment #1 werere-challenged by the intravenous inoculation of 100 fresh, pathogenic P.yoelii sporozoites. Surprisingly, all four mice that had been protectedafter a single inoculation of 10⁴ irradiated P. yoelii sporozoites withconcurrent distal IP administration of adjuvant were protected uponre-challenge. The single mouse that had been protected afteradministration of 10⁴ irradiated P. yoelii sporozoites without adjuvantwas not protected upon re-challenge. All five infectivity controlsdeveloped parasitemia (see Table 5). The protection after a singleinoculation of 10⁴ irradiated P. yoelii sporozoites with 2 μg 7DW8-5adjuvant was solidly sustained for at least 15 weeks.

TABLE 5 Example #4 - Rechallenge Experiment. Immunization with fresh,unpurified radiation attenuated Py sporozoites Re-challenge 101 days (15weeks) after 1^(st) #of fresh, 2 μg First Challenge challenge BALB/unpurified 7DW8-5 # protected/ # protected/ c mice irr PySPZ IV IP #re-challenged # re-challenged 5 1 × 10⁴ − 1/5 0/1 5 1 × 10⁴ + 4 4/4 5— + 0/5 5 Infectivity − 0/5 controls 5 Infectivity − 0/5 controls

Example 5 Single Dose Optimization

To determine the optimal single dose of attenuated sporozoites (inconjunction with 7DW8-5 concurrently administered distally IP) thatconfers protection by the IV route, mice were injected IV with 0, 2500,5000, 10,000 and 20,000 sporozoites. Two μg of 7DW8-5 were injected IP.As shown in Table 6, 80% protection was achieved in the group of micereceiving 10,000 sporozoites.

TABLE 6 Example #5 - Dose Optimization Experiment. Immunization withfresh, unpurified radiation attenuated Py sporozoites number of fresh,unpurified irr 2 μg 7DW8-5 # protected/ BALB/c mice PySPZ IV IP #challenged 5 0 + 0/5 5 0.25 × 10⁴   + 0/5 5 0.5 × 10⁴   + 2/5 5 1 ×10⁴ + 4/5 5 2 × 10⁴ + 1/5 5 Infectivity controls − 0/5

Example 6 Two Dose Optimization—Intradermal

To determine whether protective immunogenicity of attenuated sporozoitesis maintained or enhanced in the presence of 7DW8-5, mice were immunizedby intradermal (ID) injection alone, with 2 doses at 2-week intervals of15,000, 10,000, 5,000 and 2,500 sporozoites and unlike Examples 1-5, 1μg 7DW8-5 was mixed as a composition with sporozoites and thecomposition of adjuvant and sporozoites was delivered ID at the base ofthe tail. With 2 doses of 15,000 sporozoites in the presence of adjuvant100% of the mice were protected, and at lower doses of sporozoites60-80% of the mice were protected. In the absence of adjuvant, only 40%of mice receiving 2 doses of 15,000 sporozoites were protected. Thisdemonstrates that 7DW8-5 is compatible with live attenuated Plasmodiumsporozoites, and the composition enhances the protection provided byattenuated sporozoites alone.

TABLE 7 Example # 6 - Two dose Intradermal Immunization. Immunizationwith fresh, unpurified radiation attenuated Py sporozoites IDco-administered with adjuvant at base of tail. number of fresh, 1 μg #doses BALB/ unpurified 7DW8-5 per at 2-week # protected/ c mice irrPySPZ ID mouse intervals # challenged 5 1.5 × 10⁴ − two 2/5 5 1.5 ×10⁴ + two 5/5 5 1.0 × 10⁴ + two 3/5 5 0.5 × 10⁴ + two 4/5 5 0.25 ×10⁴   + two 3/5 5 0 + 0/5 8 Infectivity controls 0/8

Example 7 Single Dose Immunization Intradermal

Protection conferred by a single dose of a composition of 1 μg 7DW8-5adjuvant and varying amounts of sporozoites delivered ID was measured.For this 15,000 and 30,000 sporozoites were administered to mice ID asdescribed above. These doses were partially protective at 60% and 40%respectively.

TABLE 8 Example #7 - Single dose Intradermal Immunization Experiment.Immunization with fresh, unpurified radiation attenuated Py sporozoitesID co-administered with adjuvant at base of tail number of fresh, #doses BALB/c unpurified 1 μg7DW8-5 at 2-week # protected/ mice irr PySPZID per mouse intervals # challenged 5 1.5 × 10⁴ + one 3/5 5   3 × 10⁴ +one 2/5 5 Infectivity controls − two 0/5

Example 8 Single Dose Purified Sporozoites ID

The next step in development was to assess purified irr PySPZBALB/c/mice were immunized with irr PySPZ purified as described, in thepresence or absence of 7DW8-5 adjuvant. Three out of five (60%) micewere protected after immunization with 30,000 purified irr PySPZ mixedwith 1 μg adjuvant administered ID at the base of the tail, and nonewere protected in the absence of adjuvant. No protection was observed inmice receiving adjuvant alone.

In the foregoing, the present invention has been described withreference to suitable embodiments, but these embodiments are only forpurposes of understanding the invention and various alterations ormodifications are possible.

1-37. (canceled)
 38. A method of reducing the risk of malaria in a humanhost exposed to pathogenic Plasmodium species parasites, comprisingadministration of one or more doses of a pharmaceutical composition tosaid host prior to said exposure; wherein said pharmaceuticalcomposition comprises (i) one or more species of live attenuatedPlasmodium sporozoite-stage parasites, and (ii) an excipient; andwherein said pharmaceutical composition is co-administered with aglycolipid adjuvant represented by the structure of formula 1;

wherein R is R═(CH₂)₁₀Ph(p-F).
 39. The method of claim 38, wherein saidone or more doses comprise no more than 150,000 sporozoites.
 40. Themethod of claim 39, wherein said one or more doses comprise no more than50,000 sporozoites.
 41. The method of claim 40, wherein said one or moredoses comprise no more than 25,000 sporozoites.
 42. The method of claim38, wherein no more than 3 doses are administered.
 43. The method ofclaim 42, wherein no more than 2 doses are administered.
 44. The methodof claim 43, wherein no more than 1 dose is administered.
 45. The methodof claim 38, wherein said species are selected from the group consistingof: P. falciparum, P. vivax, P. ovale, P. knowlesi, and P. malariae. 46.The method of claim 38, wherein said species comprises P. falciparum.47. The method of claim 38, wherein said pharmaceutical composition isadministered by an intravenous, intramuscular, intradermal, orsubcutaneous route.
 48. The method of claim 47, wherein saidpharmaceutical composition is administered by an intravenous route. 49.The method of claim 38, wherein said glycolipid adjuvant is administeredby an intravenous, intramuscular, intradermal, or subcutaneous route.50. The method of claim 38, wherein the co-administration is sequential.51. The method of claim 38, wherein the co-administration is concurrent.52. The method of claim 51, wherein said glycolipid adjuvant and saidpharmaceutical composition are admixed prior to co-administration. 53.The method of claim 38, wherein said pharmaceutical composition and saidglycolipid adjuvant are co-administered by different routes ofadministration.